PROJECT SUMMARY Gut microbial metabolites have emerged as key players in human cardiometabolic health. In our pilot studies, trimethylamine N-oxide (TMAO) and equol, two diet-derived gut microbial metabolites, were associated with risk of coronary heart disease (CHD), even after adjusting for traditional CHD risk factors. These findings, in line with a growing literature on the importance of gut microbial metabolism in human cardiometabolic health, led to our hypothesis that gut microbial metabolites may be novel biomarkers and/or therapeutic targets for CHD prevention. Yet, to our knowledge, no population-based, prospective studies have applied metabolomics to systematically investigate gut microbial metabolites in relation to risk of CHD, followed by rigorous validation and evaluation of potential diet-microbiota-host interactions. Moreover, only a few studies have examined metabolomic profiles in relation to CHD risk in non-white populations, especially African Americans who bear a disproportionately high burden of CHD. For human gut microbiota research, data from populations of different races/ethnicities, geographic areas, and habitual diets are extremely valuable for determining the causality, generalizability, and clinical utility of potential biomarkers, but are currently lacking. We propose to evaluate circulating microbial metabolites in relation to risk of CHD by leveraging resources from five NIH-funded, population-based, prospective cohorts: The Southern Community Cohort Study (SCCS), Shanghai Women's and Men's Health Studies (SWMHS), Multi-Ethnic Study of Atherosclerosis (MESA), and Atherosclerosis Risk in Communities Study (ARIC). Our study has three aims: Aim 1 (discovery) will use a semi-quantitative assay that detects major groups of microbial metabolites in 900 case-control pairs of blacks, whites, and Asians (300 pairs per race) from the SCCS and SWMHS. Aim 2 (validation) will first use existing metabolomics data in the MESA and ARIC to perform an in-silico validation; confirmed metabolites will then be quantified in an independent set of 1,150 case-control pairs from SCCS, SWMHS, and MESA (~400 pairs per race). We will evaluate whether the associations vary by demographics, and whether microbial metabolites may improve CHD risk prediction. Aim 3 (diet-microbiota-host interaction) will perform a novel mediation- interaction analysis to evaluate whether and to what extent the associations are modified by dietary fiber (as prebiotics) and overall diet quality and modified or mediated by dyslipidemia, hypertension, and diabetes. This will be the first well-powered, multi-ethnic study examining gut microbial metabolites in relation to risk of CHD with careful validation, quantification, and evaluation of diet-microbiota-host interactions. This study will advance our knowledge of the role of gut microbial metabolism in CHD etiology and disparities, identify new biomarkers, and inform novel diet/microbiota-based interventions. Our team has extensive experience in metabolomics and diet-microbiota-CHD research, and thus, is uniquely positioned to accomplish this study.